1. Field of the Invention
The present invention relates to standards useful in assaying for cholesterol concentrations, and to the preparation thereof.
2. Description of the Prior Art
The use of serum standards or references in biochemistry analysis is well known. It is frequently advantageous, for example, to determine the level of certain constituents in the blood of a patient as a diagnostic aid. Serum standards are used in analytic procedures to provide a reference to which the patient's serum may be compared. Serum standards therefore may be required to have varying concentrations to certain components. For convenience, serum standards are commonly stored as a dry powder after lyophilization to be reconstituted at the time of use, or they are frozen and subsequently thawed for use. It is desirable that the serum standard be stable and have substantial optical clarity to minimize interference with the analytical measurement of serum constituents.
The prior art is believed to be best set forth in U.S. Pat. No. 3,853,465, issued to Rush et al. on Dec. 10, 1974, which is hereby incorporated by reference. This patent generally sets forth the facts of difficulties in photometric analysis which result from turbidity in serum and plasma samples. This is believed to be due primarily to the presence in the serum standard of certain serum proteins, particularly the low-density, triglyceride-rich lipoproteins. The problem of turbidity correspondingly increases when a serum standard is required to have increased triglyceride or cholesterol levels due to the consequent increase in the concentration of the turbiditycausing components.
The solution to the turbidity problem which Rush et al. set forth has generally been to add a surfactant to the serum or plasma to reduce the turbidity. Rush et al. disclose a surfactant of polyoxyethylated lauric acid having from 9 to 20 ethoxy groups. Compounds of this general class which have from 10 to about 20 ethoxy groups are disclosed in U.S. Pat. No. 3,260,648, issued to Fox on July 12, 1966, for use as an emulsifier for cholesterol in serum. This latter patent sets forth the use of a lower alkylphenoxypolyethoxyethanol having from about 10 to 20 ethoxy groups. While the use of such surfactants does result in a reduction in the turbidity of the serum, the products produced thereby tend to be metastable and separate after a period of time. Additionally, large amounts of surfactant are generally required and the use of such large amounts of surfactant may interfere with biological assays of serum ingredients. Moreover, even with the surfactants of this type, reconstitution of lyophilized serum having elevated cholesterol levels produces significant, measureable turbidity notwithstanding the use of the surfactant.
Aside from the problem of turbidity, it is recognized, as mentioned previously, that it is sometimes desirable to have a serum standard which has increased levels of triglycerides or cholesterol. In U.S. Pat. No. 3,764,556, issued to Kuchmak et al. on Oct. 9, 1973, there is disclosed a procedure for obtaining a cholesterol-rich protein fraction from outdated human plasma. The use of this protein fraction, however, entails several disadvantages. First, there are certain dangers involved in that the procedure by which the cholesterol-rich fraction is obtained will also result in collection and concentration of any hepatitis virus which may be present in the plasma. Additionally, it has found that substantial serum turbidity will result upon lyophilization and reconstitution of such a prepared cholesterol standard. Also, a process which utilizes human blood as a source of cholesterol to be added in preparing a serum standard will generally be expensive.
In an article entitled "Steroid-Protein Conjugates," by Erlanger et al., Journal of Biological Chemistry, Volume 228, September, 1957, there is disclosed the preparation of water soluble conjugates of bovine serum albumin with testosterone and with cortisone. The steroids are linked by amide bonds to the lysine residues of the albumin. The conjugates disclosed in the Erlanger et al. article were proposed as antigenic compounds which could elicit antibodies having antihormonal properties. These conjugates were proposed to be useful in enabling the development and study of antihormonal principles.